Humoral immune responses to peptides derived from the β-amyloid peptide C-terminal sequence

There is a continuing interest in the immunochemical quantification of isoforms of amyloid beta-peptide (A beta) in body fluids of patients with Alzheimer's disease (AD); however, at present there is no general procedure to produce and test the required antibodies. We examined various methods to generate rabbit anti-A beta; antibodies that are specific for A beta(38), A beta(40) and A beta(42), and we tested their specificity and sensitivity by ELISA and Western blotting. To produce high-affinity antibodies required repeated inoculations of small doses of peptide conjugates over a period of at least 6 months. Antibodies generated to peptides derived from the A beta(42) sequence showed some cross-re activity with A beta(42), but antibodies generated to A beta(4) peptides did not cross-react with A beta 42. The shortest peptide capable of generating antibodies of moderate affinity possessed the sequence Met(35)-Ala(42); however, antibodies raised to the peptide Gly(33)-Ala(42) possessed the greatest affinity (K-D = 1 nM) and specificity for A beta(42). The latter antibodies were over 50,000-fold more reactive with A beta(42) than with A beta(40). They can detect A(beta) isoforms in extracts of normal brain, where the peptides are present at levels below one part per billion. Our results provide methods to generate and characterize the specificity and affinity of anti-A beta antibodies. This information is necessary to develop sensitive and specific immunoassays to quantify AP isoforms in brain extracts and in body fluids.